Bottom Line:
Another subtype results from a reduction of actin and forms a more stable cytoplasmic body.In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization.In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere.

ABSTRACTNemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.

Fig13: Fluorescence recovery after photobleaching (FRAP) analyses of ACTA1 and ACTA1D286G. a Confocal images of ACTA1-eGFP localization at the Z-disk (white boxes) and along the thin filament (yellow boxes) in single muscle fibers of Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high embryos at 2 dpf. Image sequence shows ACTA1-eGFP prior to photobleaching (pre-bleach), at the time of photobleaching (bleach, 0 s), and 60, 300 and 600 s following photobleaching. Prior to photobleaching eGFP in Tg(ACTA1wildtype-eGFP)high muscle is primarily localized to the Z-disk (white boxes), whereas in Tg(ACTA1D286G-eGFP)high fibers, eGFP expression is more diffuse throughout the filament (yellow boxes). b Quantification of the fluorescence intensity at the Z-disk compared to the filament in Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high muscle fibers. Error bars represent SD for 12 animals (quantifying 2 fibers per animal), ***p < 0.001. c Recovery profiles for ACTA1-eGFP and ACTA1D286G-eGFP at the Z-disk and filament. Error bars represent SD for 8–10 animals (quantifying 2 fibers per animal)

Mentions:
We showed that an imbalance of actin levels in the skeletal muscle results in aggregates and muscle weakness. However, we also wanted to determine whether overexpression of ACTA1-eGFPD286G has an additional, mutation-specific, effect in the skeletal muscle. Previous studies have suggested that ACTA1D286G may have a reduced ability to polymerize and be incorporated into the sarcomere [7, 49]. To examine the dynamics of ACTA1D286G in Tg(ACTA1D286G-eGFP)high zebrafish, we performed FRAP analysis on both the thin filament and the Z-disk at 2 dpf. We found a significant reduction in the average time taken for 50 % recovery at both locations following photobleaching for ACTA1D286G-eGFP (filament: 22.74 ± 10.22 s, Z-disk: 52.38 ± 9.19 s) compared to Tg(ACTA1wildtype-eGFP)high fish (filament: 106.7 ± 34.6 s,: Z-disk: 85.79 ± 16.71 s) (Fig. 13c). This showed that the ACTA1D286G protein is readily incorporated into the sarcomere, but is more rapidly exchanged at both the Z-disk and thin filament than ACTA1wildtype, suggesting it may be less stably associated.Fig. 13

Fig13: Fluorescence recovery after photobleaching (FRAP) analyses of ACTA1 and ACTA1D286G. a Confocal images of ACTA1-eGFP localization at the Z-disk (white boxes) and along the thin filament (yellow boxes) in single muscle fibers of Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high embryos at 2 dpf. Image sequence shows ACTA1-eGFP prior to photobleaching (pre-bleach), at the time of photobleaching (bleach, 0 s), and 60, 300 and 600 s following photobleaching. Prior to photobleaching eGFP in Tg(ACTA1wildtype-eGFP)high muscle is primarily localized to the Z-disk (white boxes), whereas in Tg(ACTA1D286G-eGFP)high fibers, eGFP expression is more diffuse throughout the filament (yellow boxes). b Quantification of the fluorescence intensity at the Z-disk compared to the filament in Tg(ACTA1D286G-eGFP)high and Tg(ACTA1wildtype-eGFP)high muscle fibers. Error bars represent SD for 12 animals (quantifying 2 fibers per animal), ***p < 0.001. c Recovery profiles for ACTA1-eGFP and ACTA1D286G-eGFP at the Z-disk and filament. Error bars represent SD for 8–10 animals (quantifying 2 fibers per animal)

Mentions:
We showed that an imbalance of actin levels in the skeletal muscle results in aggregates and muscle weakness. However, we also wanted to determine whether overexpression of ACTA1-eGFPD286G has an additional, mutation-specific, effect in the skeletal muscle. Previous studies have suggested that ACTA1D286G may have a reduced ability to polymerize and be incorporated into the sarcomere [7, 49]. To examine the dynamics of ACTA1D286G in Tg(ACTA1D286G-eGFP)high zebrafish, we performed FRAP analysis on both the thin filament and the Z-disk at 2 dpf. We found a significant reduction in the average time taken for 50 % recovery at both locations following photobleaching for ACTA1D286G-eGFP (filament: 22.74 ± 10.22 s, Z-disk: 52.38 ± 9.19 s) compared to Tg(ACTA1wildtype-eGFP)high fish (filament: 106.7 ± 34.6 s,: Z-disk: 85.79 ± 16.71 s) (Fig. 13c). This showed that the ACTA1D286G protein is readily incorporated into the sarcomere, but is more rapidly exchanged at both the Z-disk and thin filament than ACTA1wildtype, suggesting it may be less stably associated.Fig. 13

Bottom Line:
Another subtype results from a reduction of actin and forms a more stable cytoplasmic body.In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization.In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere.

ABSTRACTNemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.